Central nervous system disorders have caused serious public health concerns. The number of patients in Japan with Alzheimer's disease alone, which results in cognitive dysfunction due to the degeneration and loss of nerve cells, is estimated to be more than 600,000. Currently, central nervous system disorders are treated mostly by systemic administration of therapeutic drugs. In systemic administration, however, drugs are usually incapable of passing through the blood-brain barrier and are often inefficient. Thus, many potentially useful therapeutic proteins, etc. cannot be administered systemically.
There are known methods for using an adeno-associated virus (AAV) as a vector derived from a virus for gene therapy (e.g., WO2003/018821, WO2003/053476, WO2007/001010, etc.). However, when gene transfer to a nervous system cell such as in the brain is attempted, it is necessary to consider problems including defensive functions such as the blood-brain barrier, etc., the transduction efficiency to nervous system cells, the expression efficiency, a safer route for administration, and the like.
Nakai H., et al. (Unrestricted hepatocyte transduction with adeno-associated virus serotype 8 vectors in mice. J. Virol. 2005 January; 79(1): 214-24) discloses an example of using a serotype 8 AAV vector AAV8-EF1α (-nlslacZ) expressing a LacZ gene marker with an EF1α promoter, for the purpose of gene transduction to hepatocytes.
Foust K. D., et al. (Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat. Biotechnol. 2009 January; 27(1): 59-65) discloses a self-complementary (sc) vector with the coat protein of serotype 9 AAV (AAV9) that expresses a green fluorescent protein (GFP) under the control of a chicken-β-actin hybrid promoter (CB). Duque S., et al. (Intravenous Administration of Self-complementary AAV9 Enables Transgene Delivery to Adult Motor Neurons. Mol. Ther. 2009 July; 17(7): 1187-96) also discloses a self-complementary vector (scAAV9-GFP) with a serotype 9 AAV (AAV9) capsid protein that expresses GFP under the control of the cytomegalovirus immediate-early promoter (CMV) (cf., Table 1 for the summary of the results).
Gene transfer to the brain (including neurons from neonates, astrocytes from adults, etc.) has been performed through intravascular administration of these recombinant AAV9 vectors. However, it is necessary to incorporate a reverse sequence to generate an sc type viral genome, a gene that can be incorporated into the viral genome becomes half as long as a non-sc type viral genome. Specifically, the length of the gene that can be incorporated in the sc type vector is limited to a length as small as 2 kb including the promoter and poly(A) region. By means of this limitation, therapeutic applications of recombinant viral vectors are limited as well.
As described above, various recombinant adeno-associated virus vectors have been produced. However, there are unknown vectors such recombinant AAV vectors that can make use of non-sc form AAV genomes that are capable of passing through the blood-brain barrier in a living subject and as associated with simple administration to enable efficient gene transfer especially to a nervous system cell in the brain, whereby a wider range of therapeutic applications can be expected.